Methods and means for producing efficient silencing construct using recombinational cloning

ABSTRACT

Methods and vectors and kits are provided for producing chimeric nucleic acid constructs capable of producing dsRNA for silencing target nucleic acid sequences of interest using recombinational cloning.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. application Ser. No.10/055,001, filed Jan. 25, 2002, and from U.S. Provisional ApplicationSer. No. 60/264, 067, filed Jan. 25, 2001 and U.S. ProvisionalApplication Ser. No. 60/333,743, filed Nov. 29, 2001. The entire contentof these prior applications is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to efficient methods and means for producingchimeric nucleic acid constructs capable of producing dsRNA useful forsilencing target nucleic acid sequences of interest. The efficiency ofthe disclosed methods and means further allows high throughput analysismethods to determine the function of isolated nucleic acids, such asESTs, without a known function and may further be put to use to isolateparticular genes or nucleotide sequences from a preselected group ofgenes.

BACKGROUND ART

Increasingly, the nucleotide sequence of whole genomes of organisms,including Arabidopsis thaliana, has been determined and as these databecome available, they provide a wealth of unmined information. Theultimate goal of these genome projects is to identify the biologicalfunction of every gene in the genome.

Attribution of a function to a nucleic acid with a particular nucleotidesequence can be achieved in a variety of ways. Some of the genes havebeen characterized directly using the appropriate assays. Others havebeen attributed with a tentative function through homology with (partsof) genes having a known function in other organisms. Loss-of-functionmutants, obtained e.g. by tagged insertional mutagenesis have also beenvery informative about the role of some of these unknown genes(AzpiroLeehan and Feldmann 1997; Martienssen 1998) particularly in thelarge-scale analysis of the yeast genome (Ross-MacDonald et al., 1999).

Structural mutants resulting in a loss-of-function may also be mimickedby interfering with the expression of a nucleic acid of interest at thetranscriptional or post-transcriptional level. Silencing of genes,particularly plant genes using anti-sense or co-suppression constructsto identify gene function, especially for a larger number of targets, ishowever hampered by the relatively low proportion of silencedindividuals obtained, particularly those wherein the silencing level isalmost complete.

Recent work has demonstrated that the silencing efficiency could begreatly improved both on quantitative and qualitative level usingchimeric constructs encoding RNA capable of forming a double strandedRNA by basepairing between the antisense and sense RNA nucleotidesequences respectively complementary and homologous to the targetsequences.

Fire et al., 1998 describe specific genetic interference by experimentalintroduction of double-stranded RNA in Caenorhabditis elegans. Theimportance of these findings for functional genomics has been discussed(Wagner and Sun, 1998).

WO 99/32619 provides a process of introducing RNA into a living cell toinhibit gene expression of a target gene in that cell. The process maybe practiced ex vivo or in vivo. The RNA has a region withdouble-stranded structure. Inhibition is sequence-specific in that thenucleotide sequences of the duplex region of the RNA and or a portion ofthe target gene are identical.

Waterhouse et al. 1998 describes that virus resistance and genesilencing in plants can be induced by simultaneous expression of senseand anti-sense RNA. The sense and antisense RNA may be located in onetranscript that has self-complementarity.

Hamilton et al. 1998 describes that a transgene with repeated DNA, i.e.inverted copies of its 5′ untranslated region, causes high frequency,post-transcriptional suppression of ACC-oxidase expression in tomato.

WO 98/53083 describes constructs and methods for enhancing theinhibition of a target gene within an organism, which involve insertinginto the gene-silencing vector an inverted, repeat sequence of all orpart of a polynucleotide region within the vector.

WO 99/53050 provides methods and means for reducing the phenotypicexpression of a nucleic acid of interest in eukaryotic cells,particularly in plant cells. These methods involve introducing chimericgenes encoding sense and antisense RNA molecules directed towards thetarget nucleic acid, which are capable of forming a double stranded RNAregion by base-pairing between the regions with the sense and antisensenucleotide sequence, or introducing the RNA molecules themselves.Preferably, the RNA molecules comprise simultaneously both sense andantisense nucleotide sequences.

WO 99/49029 relates generally to a method of modifying gene expressionand to synthetic genes for modifying endogenous gene expression in acell, tissue or organ of a transgenic organism, in particular to atransgenic animal of plant. Synthetic genes and genetic constructs,capable of forming a dsRNA which are capable of repressing, delaying orotherwise reducing the expression of an endogenous gene or a target genein an organism when introduced thereto are also provided.

WO 99/61631 relates to methods to alter the expression of a target genein a plant using sense and antisense RNA fragments of the gene. Thesense and antisense RNA fragments are capable of pairing and forming adouble-stranded RNA molecule, thereby altering the expression of thegene. The present invention also relates to plants, their progeny andseeds thereof obtained using these methods.

WO 00/01846 provides a method of identifying DNA responsible forconferring a particular phenotype in a cell. That method comprises a)constructing a cDNA or genomic library of the DNA of the cell in asuitable vector in an orientation relative to (a) promoter(s) capable ofinitiating transcription of the cDNA or DNA to double stranded (ds) RNAupon binding of an appropriate transcription factor to the promoter(s);b) introducing the library into one or more of cells comprising thetranscription factor, and c) identifying and isolating a particularphenotype of a cell comprising the library and identifying the DNA orcDNA fragment from the library responsible for conferring the phenotype.Using this technique, it is also possible to assign function to a knownDNA sequence by a) identifying homologues of the DNA sequence in a cell,b) isolating the relevant DNA homologue(s) or a fragment thereof fromthe cell, c) cloning the homologue or fragment thereof into anappropriate vector in an orientation relative to a suitable promotercapable of initiating transcription of dsRNA from said DNA homologue orfragment upon binding of an appropriate transcription factor to thepromoter and d) introducing the vector into the cell from step a)comprising the transcription factor.

WO 00/44914 also describes composition and methods for in vivo and invitro attenuation of gene expression using double stranded RNA,particularly in zebrafish.

WO 00/49035 discloses a method for silencing the expression of anendogenous gene in a cell. That method involves overexpressing in thecell a nucleic acid molecule of the endogenous gene and an antisensemolecule including a nucleic acid molecule complementary to the nucleicacid molecule of the endogenous gene, wherein the overexpression of thenucleic acid molecule of the endogenous gene and the antisense moleculein the cell silences the expression of the endogenous gene.

Smith et al., 2000 as well as WO 99/53050 described that introncontaining dsRNA further increased the efficiency of silencing.

However, the prior art has not solved the problems associated with theefficient conversion of any nucleotide sequence of interest into achimeric construct capable of producing a dsRNA in eukaryotic cells,particularly in plant cells, and preferably in a way amenable to theprocessing of large number of nucleotide sequences.

These and other problems have been solved as described hereinafter inthe different embodiments and claims.

SUMMARY OF THE INVENTION

It is an object of the invention to provide vectors comprising thefollowing operably linked DNA fragments a) an origin of replicationallowing replication in microorganisms (1), preferably bacteria;particularly Escherichia coli; b) a selectable marker region (2) capableof being expressed in microorganisms, preferably bacteria; and c) achimeric DNA construct comprising in sequence (i) a promoter or promoterregion (3) capable of being recognized by RNA polymerases of aeukaryotic cell, preferably a plant-expressible promoter; (ii) a firstrecombination site (4), a second recombination site (5), a thirdrecombination site (6) and a fourth recombination site (7); and (iii) a3′ transcription terminating and polyadenylation region (8) functionalin the eukaryotic cell; wherein the first recombination site (4) and thefourth recombination site (7) are capable of reacting with a samerecombination site, preferably are identical, and the secondrecombination site (5) and the third recombination site (6), are capableof reacting with a same recombination site, preferably are identical;and wherein the first recombination site (4) and the secondrecombination site (5) do not recombine with each other or with a samerecombination site or the third recombination site (6) and the fourthrecombination site (7) do not recombine with each other or with a samerecombination site. Optionally the vector may further include additionalelements such as: a second selectable marker gene (9) between the first(4) and second recombination site (5) and/or a third selectable markergene (10) between the third (6) and fourth recombination site (7) and/ora region flanked by intron processing signals (11), preferably anintron, functional in the eukaryotic cell, located between the secondrecombination site (5) and the third recombination site (6) and/or afourth selectable marker gene (19), located between the second (5) andthird recombination site (6) and/or left and right border T-DNAsequences flanking the chimeric DNA construct and/or a selectable markergene capable of being expressed in eukaryotic, preferably plant, cells,preferably located between the left and the right T-DNA border sequencesand/or an origin of replication capable of functioning in Agrobacteriumspp. Selectable marker genes may be selected from the group consistingof an antibiotic resistance gene, a tRNA gene, an auxotrophic marker, atoxic gene, a phenotypic marker, an antisense oligonucleotide; arestriction endonuclease; a restriction endonuclease cleavage site, anenzyme cleavage site, a protein binding site, an a sequencecomplementary PCR primer. Preferably the first (4) and fourthrecombination site (7) are attR1 comprising the nucleotide sequence ofSEQ ID No 4 and the second (5) and third (6) recombination site areattR2 comprising the nucleotide sequence of SEQ ID No 5 or the first (4)and fourth recombination site (7) are attP1 comprising the nucleotidesequence of SEQ ID No 10 and the second (5) and third (6) recombinationsite are attP2 comprising the nucleotide sequence of SEQ ID No 11.

It is another objective of the invention to provide a kit comprising anacceptor vector according to invention, preferably further comprising atleast one recombination protein capable of recombining a DNA segmentcomprising at least one of the recombination sites.

It is yet another objective of the invention to provide a method formaking a chimeric DNA construct capable of expressing a dsRNA in aeukaryotic cell comprising the steps of combining in vitro:

-   -   an acceptor vector as herein before described;    -   an insert DNA, preferably a linear or circular insert DNA,        comprising        -   a DNA segment of interest (12) flanked by        -   a fifth recombination site (13) which is capable of            recombining with the first (4) or fourth recombination            site (7) on the vector; and        -   a sixth recombination site (14) which is capable of            recombining with the second (5) or third recombination            site (6) on the vector;        -   at least one site specific recombination protein capable of            recombining the first (4) or fourth (7) and the fifth            recombination site (13) and the second (5) or third (6) and            the sixth recombination site (14);    -   allowing recombination to occur in the presence of at least one        recombination protein, preferably selected from Int and IHF        and (ii) Int, Xis, and IHF, so as to produce a reaction mixture        comprising product DNA molecules, the product DNA molecule        comprising in sequence:        -   the promoter or promoter region (3) capable of being            recognized by RNA polymerases of the eukaryotic cell;        -   a recombination site (15) which is the recombination product            of the first (4) and the fifth recombination site (13);        -   the DNA fragment of interest (12);        -   a recombination site (16) which is the recombination product            of the second (4) and the sixth recombination site (14);        -   a recombination site (17) which is the recombination product            of the third (5) and the sixth recombination site (14);        -   the DNA fragment of interest in opposite orientation (12);        -   a recombination site (18) which is the recombination product            of the fourth (7) and the fifth recombination site (13); and        -   the 3′ transcription terminating and polyadenylation            region (8) functional in the eukaryotic cell; and    -   selecting the product DNA molecules, preferably in vivo.

The method allows that multiple insert DNAs comprising different DNAfragments of interest are processed simultaneously.

The invention also provides a method for preparing a eukaryoticnon-human organism, preferably a plant, wherein the expression of atarget nucleic acid of interest is reduced or inhibited, the methodcomprising:

-   -   preparing a chimeric DNA construct capable of expressing a dsRNA        in cells of the eukaryotic non-human organism according to        methods of the invention;    -   introducing the chimeric DNA construct in cells of the        eukaryotic non-human organism; and isolating the transgenic        eukaryotic organism.

It is also an objective of the invention to provide a method forisolating a nucleic acid molecule involved in determining a particulartrait, comprising the steps of:

-   -   preparing a library of chimeric DNA constructs capable of        expressing a dsRNA in cells of the eukaryotic non-human organism        according to any one of the methods of the invention;    -   introducing individual representatives of the library of        chimeric DNA constructs in cells of the eukaryotic non-human        organism;    -   isolating a eukaryotic organism exhibiting the particular trait;        and isolating the nucleic acid molecule.

The invention also provides a eukaryotic non-human organism, preferablya plant comprising a chimeric DNA construct obtainable through themethods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of vectors and method used in apreferred embodiment of the invention.

FIG. 1A: A nucleic acid of interest (12) is amplified by PCR usingprimers comprising two different recombination sites (13, 14) whichcannot react with each other or with the same other recombination site.This results in “insert DNA” wherein the nucleic acid of interest (12)is flanked by two different recombination sites (13, 14).

FIG. 1B. Using at least one recombination protein, the insert DNA isallowed to recombine with the acceptor vector between the recombinationsites, whereby the first (4) and fourth recombination site (7) reactwith one of the recombination sites (13) flanking the PCR amplified DNAof interest (12) and the second (5) and third (6) recombination site onthe acceptor vector recombine with the other recombination site (14)flanking the DNA of interest (12). The desired product DNA can beisolated by selecting for loss of the selectable marker genes (9) and(10) located between respectively the first (4) and second (5)recombination sites and the third (6) and fourth (7) recombinationsites. Optionally, an additional selectable marker gene may be includedbetween the second (5) and third (6) recombination site to allowselection for the presence of this selectable marker gene andconsequently for the optional intron sequence, which is flanked byfunctional intron processing signal sequences (11). The acceptor vector,as well as the product vector further comprises an origin of replication(Ori; (1)) and a selectable marker gene (2) to allow selection for thepresence of the plasmid.

This results in a chimeric DNA construct with the desired configurationcomprising a eukaryotic promoter region (3); a recombination site (15)produced by the recombination between recombination sites (4) and (13);a first copy of the DNA of interest (12); a recombination site (16)produced by the recombination between recombination sites (5) and (14);optionally an intron sequence flanked by intron processing signals (11);a recombination site (17) produced by the recombination betweenrecombination sites (6) and (14); a second copy of the DNA of interest(12) in opposite orientation to the first copy of the DNA of interest; arecombination site (18) produced by the recombination betweenrecombination sites (7) and (13); a eukaryotic transcription terminatorand polyadenylation signal (8).

FIG. 2A: A nucleic acid of interest (12) is amplified by PCR usingprimers comprising two different recombination sites which uponrecombination with the recombination sites on an intermediate vector(FIG. 2B) will yield recombination sites compatible with the first (4)and fourth (5) and with the second (6) and third (7) recombination siteon the acceptor vector respectively.

FIG. 2B: The insert DNA obtained in FIG. 2A is allowed to recombine withthe intermediate vector in the presence of at least one recombinationprotein to obtain an intermediate DNA wherein the DNA of interest (12)is flanked by two different recombination sites (13, 14) and whichfurther comprises an origin of replication (1) and a selectable markergene (2).

FIG. 2C: The intermediate DNA is then allowed to recombine with theacceptor vector using at least one second recombination protein(basically as described for FIG. 1B).

FIG. 3: Schematic representation of the acceptor vector “pHELLSGATE”

FIG. 4: Schematic representation of the acceptor vectors “pHELLSGATE 8”“pHELLSGATE 11” and “pHELLSGATE 12”.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The current invention is based on the unexpected finding by theinventors that recombinational cloning was an efficient one-step methodto convert a nucleic acid fragment of interest into a chimeric DNAconstruct capable of producing a dsRNA transcript comprising a sense andantisense nucleotide sequence capable of being expressed in eukaryoticcells. The dsRNA molecules are efficient effectors of gene silencing.These methods improve the efficiency problems previously encountered toproduce chimeric DNAs with long inverted repeats.

Thus, in a first embodiment, the invention provides a method for makinga chimeric DNA construct or chimeric gene capable of expressing an RNAtranscript in a eukaryotic cell, the RNA being capable of internalbasepairing between a stretch of nucleotides corresponding to a nucleicacid of interest and its complement (i.e. the stretch of nucleotides ininverted orientation) located elsewhere in the transcript (and thusforming a hairpin RNA) comprising the following steps:

-   providing an “acceptor vector” comprising the following operably    linked DNA fragments:    -   an origin of replication allowing replication in a host cell        (1),    -   a selectable marker region (2) capable of being expressed in the        host cell; and    -   a chimeric DNA construct comprising in sequence:        -   a promoter or promoter region (3) capable of being            recognized by RNA polymerases of a eukaryotic cell;        -   a first recombination site (4), a second recombination site            (5), a third recombination site (6) and a fourth            recombination site (7), whereby            -   the first (4) and fourth recombination site (7) are                capable of reacting with the same other recombination                site and preferably are identical to each other;            -   the second (5) and third (6) recombination site are also                capable of reacting with the same other recombination                site and preferably are identical to each other;            -   the first (4) and second (5) recombination site do not                recombine with each other or with the same other                recombination site; and            -   the third (6) and fourth (7) recombination site do not                recombine with each other or with the same other                recombination site; and    -   a 3′ transcription terminating and polyadenylation region (8)        functional in a eukaryotic cell;-   providing an “insert DNA” comprising    -   the DNA segment of interest (12) flanked by    -   a fifth recombination site (13) which is capable of recombining        with the first(4) or fourth (7) recombination site but        preferably not with the second (5) or third (6) recombination        site;    -   a sixth recombination site (14), which is capable of recombining        with the second (5) or third (6) recombination site but        preferably not with the first (4) or fourth (7) recombination        site.-   combining in vitro the insert DNA and the acceptor vector in the    presence of at least one specific recombination protein; and-   allowing the recombination to occur to produce a reaction mixture    comprising inter alia “product DNA” molecules which comprise in    sequence    -   the promoter or promoter region (3) capable of being recognized        by RNA polymerases of a eukaryotic cell;    -   a recombination site (15) which is the recombination product of        the first (4) and fifth recombination site (13);    -   a first copy of the DNA fragment of interest (12);    -   a recombination site (16) which is the recombination product of        the second (4) and the sixth recombination site (14);    -   a recombination site (17) which is the recombination product of        the third (5) and the sixth recombination site (14);    -   a second copy of the DNA fragment of interest in opposite        orientation (12) with regard to the first copy;    -   a recombination site (18) which is the recombination product of        the fourth (7) and the fifth recombination site (13); and    -   a 3′ transcription terminating and polyadenylation region (8)        functional in a eukaryotic cell; and-   selecting the product DNA molecules.    This method is schematically outlined in FIG. 1, with non-limiting    examples of recombination sites and selectable markers.

As used herein, a “host cell” is any prokaryotic or eukaryotic organismthat can be a recipient for the acceptor vector or the product DNA.Conveniently, the host cell will be an Escherichia coli strain commonlyused in recombinant DNA methods.

A “recombination protein” is used herein to collectively refer tosite-specific recombinases and associated proteins and/or co-factors.Site-specific recombinases are enzymes that are present in some virusesand bacteria and have been characterized to have both endonuclease andligase properties. These recombinases (along with associated proteins insome cases) recognize specific sequences of bases in DNA and exchangethe DNA segments flanking those segments. Various recombination proteinsare described in the art(see WO 96/40724 herein incorporated byreference in its entirety, at least on page 22 to 26). Examples of suchrecombinases include Cre from bacteriophage P1 and Integrase frombacteriophage lambda.

Cre is a protein from bacteriophage P1 (Abremski and Hoess, 1984) whichcatalyzes the exchange between 34 bp DNA sequences called loxP sites(see Hoess et al., 1986. Cre is available commercially (Novagen, Catalog69247-1).

Integrase (Int) is a protein from bacteriophage lambda that mediates theintegration of the lambda genome into the E. coli chromosome. Thebacteriophage lambda Int recombinational proteins promote irreversiblerecombination between its substrate att sites as part of the formationor induction of a lysogenic state. Reversibility of the recombinationreactions results from two independent pathways for integrative orexcisive recombination. Cooperative and competitive interactionsinvolving four proteins (Int, Xis, IHF and FIS) determine the directionof recombination. Integrative recombination involves the Int and IHFproteins and attP (240 bp) and attB (25b) recombination sites.Recombination results in the formation of two new sites: attL and affR.A commercial preparation comprising Int and IHF proteins is commerciallyavailable (BP clonase™; Life Technologies). Excisive recombinationrequires Int, IHF, and Xis and sites attL and attR to generate attP andattB. A commercial preparation comprising Int, IHF and Xis proteins iscommercially available (LR clonase™; Life Technologies).

A “recombination site” as used herein refers to particular DNAsequences, which a recombinase and possibly associated proteinsrecognizes and binds. The recombination site recognized by Crerecombinase is loxP which is a 34 base pair sequence comprised of two 13base pair inverted repeats (serving as recombinase binding sites)flanking an 8 base pair core sequence. The recombination sites attB,attP, attL and attR are recognized by lambda integrase. AttB is anapproximately 25 base pair sequence containing two 9 base pair core-typeInt binding sites and a 7 base pair overlap region. AttP is anapproximately 240 base pair sequence containing core-type Int bindingsites and arm-type Int binding sites as well as sites for auxiliaryproteins IHF, FIS and Xis (Landy 1993). Each of the att sites contains a15 bp core sequence with individual sequence elements of functionalsignificance lying within, outside and across the boundaries of thiscommon core (Landy, 1989) Efficient recombination between the variousatt sites requires that the sequence of the central common region issubstantially identical between the recombining partners. The exactsequence however is modifiable as disclosed in WO 96/40724 and thevariant recombination sites selected from

attB1: AGCCTGCTTTTTTGTACAAACTTGT; (SEQ ID No 1) attB2:AGCCTGCTTTCTTGTACAAACTTGT; (SEQ ID No 2) attB3:ACCCAGCTTTCTTGTACAAACTTGT; (SEQ ID No 3) attR1:GTTCAGCTTTTTTGTACAAACTTGT; (SEQ ID No 4) attR2:GTTCAGCTTTCTTGTACAAACTTGT; (SEQ ID No 5) attR3:GTTCAGCTTTCTTGTACAAAGTTGG; (SEQ ID No 6) attL1:AGCCTGCTTTTTTGTACAAAGTTGG; (SEQ ID No 7) attL2:AGCCTGCTTTCTTGTACAAAGTTGG; (SEQ ID No 8) attL3:ACCCAGCTTTCTTGTACAAAGTTGG; (SEQ ID No 9) attP1:GTTCAGCTTTTTTGTACAAAGTTGG; (SEQ ID No 10) or attP2,P3:GTTCAGCTTTCTTGTACAAAGTTGG (SEQ ID No 11)allow more flexibility in the choice of suitable pairs or recombinationsites that have the capability to recombine (as indicated by their indexnumber).

It will be clear to the skilled artisan that a correspondence isrequired between the recombination site(s) used and the recombinationproteins used.

In one embodiment, the following combinations of recombination sites forthe acceptor vector are present in the acceptor vector:

-   -   the first (4) and fourth (7) recombination sites are identical        and comprise        -   attP1 comprising            -   the nucleotide sequence of SEQ ID No 10 and    -   the second (5) and third (6) recombination site are also        identical and comprise        -   attP2 comprising            -   the nucleotide sequence of SEQ ID No 11; or    -   the first (4) and fourth (7) recombination sites are identical        and comprise        -   attR1 comprising            -   the nucleotide sequence of SEQ ID No 4 and    -   the second (5) and third (6) recombination site are also        identical and comprise        -   attR2 comprising            -   the nucleotide sequence of SEQ ID No 5; and                the following combinations of recombination sites for                the insert DNA are used:    -   the fifth (13) recombination site comprises        -   attB1 comprising            -   the nucleotide sequence of SEQ ID No 1 and    -   the sixth (14) recombination site comprises        -   attB2 comprising            -   the nucleotide sequence of SEQ ID No 2,                the combination being suitable for recombination with                the first acceptor vector mentioned above; or    -   the fifth (13) recombination site comprises        -   attL1 comprising            -   the nucleotide sequence of SEQ ID No 7 and    -   the sixth (14) recombination site comprises        -   attL2 comprising            -   the nucleotide sequence of SEQ ID No 8,                the combination being suitable for recombination with                the second acceptor vector mentioned above.

It has been unexpectedly found that product DNA molecules (resultingfrom recombination between the above mentioned second acceptor vectorwith attR recombination sites (such as pHELLSGATE 8) and insert DNAflanked by attL recombination sites) wherein the gene inserts in bothorientations are flanked by attB recombination sites are more effectivein silencing of the target gene(both quantitatively and qualitatively)than product DNA molecules (resulting from recombination between theabove mentioned first acceptor vector with attP recombination sites(such as pHELLSGATE or pHELLSGATE 4) and insert DNA flanked by attBrecombination sites) wherein the gene inserts in both orientations areflanked by attL recombination sites. Although not intending to limit theinvention to a particular mode of action it is thought that the greaterlength of the attL sites and potential secondary structures therein mayact to inhibit transcription yielding the required dsRNA to a certainextent. However, acceptor vectors such as the above mentioned firstacceptor vectors with attP sites may be used when target gene silencingto a lesser extent would be useful or required.

The dsRNA obtained by the chimeric DNA construct made according to theinvention may be used to silence a nucleic acid of interest, i.e., toreduce its phenotypic expression, in a eukaryotic organism, particularlya plant, either directly or by transcription of the chimeric DNAconstruct in the cells of the eukaryotic organism. When this is thecase, the following considerations may apply.

The length of the nucleic acid of interest (12) may vary from about 10nucleotides (nt) up to a length equaling the length (in nucleotides) ofthe target nucleic acid whose phenotypic expression is to be reduced.Preferably the total length of the sense nucleotide sequence is at least10 nt, or at least 19 nt, or at least 21 nt, or at least 25 nt, or atleast about 50 nt, or at least about 100 nt, or at least about 150 nt,or at least about 200 nt, or at least about 500 nt. It is expected thatthere is no upper limit to the total length of the sense nucleotidesequence, other than the total length of the target nucleic acid.However for practical reasons (such as, e.g., stability of the chimericgenes) it is expected that the length of the sense nucleotide sequenceshould not exceed 5000 nt, particularly should not exceed 2500 nt andcould be limited to about 1000 nt.

It will be appreciated that the longer the total length of the nucleicacid of interest (12), the less stringent the requirements for sequenceidentity between the nucleic acid of interest and the correspondingsequence in the target gene. Preferably, the nucleic acid of interestshould have a sequence identity of at least about 75% with thecorresponding target sequence, particularly at least about 80%, moreparticularly at least about 85%, quite particularly about 90%,especially about 95%, more especially about 100%, quite especially beidentical to the corresponding part of the target nucleic acid. However,it is preferred that the nucleic acid of interest always includes asequence of about 10 consecutive nucleotides, particularly about 25 nt,more particularly about 50 nt, especially about 100 nt, quite especiallyabout 150 nt with 100% sequence identity to the corresponding part ofthe target nucleic acid. Preferably, for calculating the sequenceidentity and designing the corresponding sense sequence, the number ofgaps should be minimized, particularly for the shorter sense sequences.

For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970). The computer-assisted sequence alignment above, can beconveniently performed using standard software program such as GAP,which is part of the Wisconsin Package Version 10.1 (Genetics ComputerGroup, Madison, Wis., USA) using the default scoring matrix with a gapcreation penalty of 50 and a gap extension penalty of 3. Sequences areindicated as “essentially similar” when such sequence have a sequenceidentity of at least about 75%, particularly at least about 80%, moreparticularly at least about 85%, quite particularly about 90%,especially about 95%, more especially about 100%, quite especially areidentical. It is clear than when RNA sequences are the to be essentiallysimilar or have a certain degree of sequence identity with DNAsequences, thymine (T) in the DNA sequence is considered equal to uracil(U) in the RNA sequence.

The “insert DNA” may conveniently be provided using DNA amplificationprocedures, such as PCR, of the nucleic acid of interest, using asprimers oligonucleotide sequences incorporating appropriaterecombination sites as well as oligonucleotide sequences appropriate forthe amplification of the nucleic acid of interest. However, alternativemethods are available in the art to provide the nucleic acid of interestwith the flanking recombination sites, including but not limited tocovalently linking oligonucleotides or nucleic acid fragments comprisingsuch recombination sites to the nucleic acid(s) of interest usingligase(s).

The providing of the appropriate flanking recombination sites to thenucleic acid may also proceed in several steps. For example, in a firststep the flanking sites provided to the nucleic acid of interest may besuch that upon recombination with the recombination sites in anintermediate vector new recombination sites are created flanking thenucleic acid of interest, now compatible for recombination with theacceptor vector. This scheme is outlined in FIG. 2, with non-limitingexamples of recombination sites and selectable markers. It is understoodthat the insert DNA may be in a circular form or in a linear form.

As used herein, an “origin of replication” is a DNA fragment whichallows replication of the acceptor vector in microorganisms, preferablybacteria, particularly E. coli strains, and ensures that uponmultiplication of the microorganism, the daughter cells receive copiesof the acceptor vector.

“Selectable marker (gene)” is used herein to indicate a DNA segment thatallows selection or screening for the presence or absence of that DNAsegment under suitable conditions. Selectable markers include but arenot limited to:

DNA segments that encode products which provide resistance againstotherwise toxic compounds (e.g. antibiotic resistance genes, herbicideresistance genes);

DNA segments encoding products which are otherwise lacking in therecipient cell (e.g. tRNA genes, auxotrophic markers);

DNA segments encoding products which suppress the activity of a geneproduct;

DNA segments encoding products which can readily be identified (e.g.β-galactosidase, green fluorescent protein (GFP), β-glucuronidase(GUS));

DNA segments that bind products which are otherwise detrimental to cellsurvival and/or function;

DNA segments that are capable of inhibiting the activity of any of theDNA segments Nos (1) to (5) (e.g. antisense oligonucleotides);

DNA segments that bind products that modify a substrate (e.g.restriction endonuclease);

DNA segments that can be used to isolate a desired molecule (e.g.specific protein binding sites);

DNA segments that encode a specific nucleotide sequence which can beotherwise non-functional (e.g. for PCR amplification of subpopulationsof molecules);

DNA segments, which when absent, directly or indirectly confersensitivity to particular compound(s); and/or

DNA segments, which when absent, directly or indirectly conferresistance to particular compound(s).

Preferred first selectable markers (2) are antibiotic resistance genes.A large number of antibiotic resistance genes, particularly which can beused in bacteria, are available in the art and include but are notlimited to aminoglycoside phosphotransferase I and II, chloramphenicolacetyltransferase, beta-lactamase, and/or aminoglycosideadenosyltransferase.

Preferred second selectable markers (9) and third selectable markers(10) are selectable markers allowing a positive selection when absent ordeleted after recombination (i.e. in the product DNA) such as but notlimited to ccdB gene the product of which interferes with E coli DNAgyrase and thereby inhibits growth of most E. coli strains. Preferably,the second and third markers are identical.

In one embodiment of the invention, the acceptor comprises a fourthselectable marker (19) between the second (5) and third (6)recombination site, preferably a marker allowing positive selection forthe presence thereof, such as a antibiotic resistance gene, e.g.chloramphenicol resistance gene. Preferably, the fourth selectablemarker should be different from first selectable marker and differentfrom the second and third selectable marker. The presence of a fourthselectable marker allows to select or screen for the retention of theDNA region between the second (5) and third (6) recombination site inthe product DNA. This increases the efficiency with which the desiredproduct DNAs having the nucleic acid of interest cloned in invertedrepeat and operably linked to eukaryotic expression signals may beobtained. However, it has been found that with most of the acceptorvectors tested, the presence of a selectable marker is not required andhas little influence on the ratio of expected and desired product DNAmolecules (which usually exceeds about 90% of obtained product DNAmolecules) to undesired product DNA molecules.

It will be understood that a person skilled in the art has a number oftechniques available for recognizing the expected and desired productDNA molecules, such as but not limited to restriction enzyme digests oreven determining the nucleotide sequence of the recombination product.

In another embodiment of the invention, the acceptor vector furthercomprises a pair of intron processing signals (11) or an intron sequencefunctional in the eukaryotic cell, preferably located between the second(5) and third (6) recombination site. However, the pair of intronprocessing signals or the intron may also be located elsewhere in thechimeric construct between the promoter or promoter region (3) and theterminator region (8). As indicated in the background art, this willimprove the efficiency with which the chimeric DNA construct encodingthe dsRNA will be capable of reducing the phenotypic expression of thetarget gene in the eukaryotic cell. A particularly preferred intronfunctional in cells of plants is the pdk intron (Flaveria trinerviapyruvate orthophosphate dikinase intron 2; see WO99/53050 incorporatedby reference). The fourth selectable marker (19) may be located betweenthe intron processing signals or within the intron (if these are locatedbetween the second and third recombination site), but may also belocated adjacent to the intron processing signals or the intron.

A person skilled in the art will recognize that the product DNAmolecules, resulting from a recombination with an acceptor vector asherein described, which comprise a region between the second (5) andthird (6) recombination will fall into two classes which can berecognized by virtue of the orientation of that intervening region. Inthe embodiments wherein the acceptor vector also comprises an intron,the different orientation may necessitate an additional step ofidentifying the correct orientation. To avoid this additional step, theacceptor vector may comprise an intron that can be spliced outindependent of its orientation (such as present in pHELLSGATE 11) or theacceptor vector may comprise a spliceable intron in both orientations(such as present in pHELLSGATE 12).

As used herein, the term “promoter” denotes any DNA that is recognizedand bound (directly or indirectly) by a DNA-dependent RNA-polymeraseduring initiation of transcription. A promoter includes thetranscription initiation site, and binding sites for transcriptioninitiation factors and RNA polymerase, and can comprise various othersites (e.g., enhancers), at which gene expression regulatory proteinsmay bind.

The term “regulatory region”, as used herein, means any DNA that isinvolved in driving transcription and controlling (i.e., regulating) thetiming and level of transcription of a given DNA sequence, such as a DNAcoding for a protein or polypeptide. For example, a 5′ regulatory region(or “promoter region”) is a DNA sequence located upstream (i.e., 5′) ofa coding sequence and which comprises the promoter and the5′-untranslated leader sequence. A 3′ regulatory region is a DNAsequence located downstream (i.e., 3′) of the coding sequence and whichcomprises suitable transcription termination (and/or regulation)signals, including one or more polyadenylation signals.

As used herein, the term “plant-expressible promoter” means a DNAsequence that is capable of controlling (initiating) transcription in aplant cell. This includes any promoter of plant origin, but also anypromoter of non-plant origin which is capable of directing transcriptionin a plant cell, i.e., certain promoters of viral or bacterial originsuch as the CaMV35S, the subterranean clover virus promoter No 4 or No7, or T-DNA gene promoters. Other suitable promoters includetissue-specific or organ-specific promoters including but not limited toseed-specific promoters (e.g., WO89/03887), organ-primordia specificpromoters (An et al., 1996), stem-specific promoters (Keller et al.,1988), leaf specific promoters (Hudspeth et al., 1989),mesophyl-specific promoters (such as the light-inducible Rubiscopromoters), root-specific promoters (Keller et al., 1989),tuber-specific promoters (Keil et al., 1989), vascular tissue specificpromoters (Peleman et al., 1989), stamen-selective promoters (WO89/10396, WO 92/13956), dehiscence zone specific promoters (WO 97/13865)and the like.

The acceptor vector may further comprise a selectable marker forexpression in a eukaryotic cell. Selectable marker genes for expressionin eukaryotic cells are well known in the art, including but not limitedto chimeric marker genes. The chimeric marker gene can comprise a markerDNA that is operably linked at its 5′ end to a promoter, functioning inthe host cell of interest, particularly a plant-expressible promoter,preferably a constitutive promoter, such as the CaMV ³⁵S promoter, or alight inducible promoter such as the promoter of the gene encoding thesmall subunit of Rubisco; and operably linked at its 3′ end to suitableplant transcription 3′ end formation and polyadenylation signals. It isexpected that the choice of the marker DNA is not critical, and anysuitable marker DNA can be used. For example, a marker DNA can encode aprotein that provides a distinguishable color to the transformed plantcell, such as the A1 gene (Meyer et al., 1987), can provide herbicideresistance to the transformed plant cell, such as the bar gene, encodingresistance to phosphinothricin (EP 0,242,246), or can provide antibioticresistance to the transformed cells, such as the aac(6′) gene, encodingresistance to gentamycin (WO94/01560).

The acceptor vector may also further comprise left and right T-DNAborder sequences flanking the chimeric DNA construct, and may comprisean origin of replication functional in Agrobacterium spp. and/or a DNAregion of homology with a helper Ti-plasmid as described in EP0 116718.

The efficiency and ease by which any nucleic acid of interest may beconverted into a chimeric DNA construct comprising two copies of thenucleic acid of interest in inverted repeat and operably linked toeukaryotic 5′ and 3′ regulatory regions using the means and methodsaccording to the invention, makes these particularly apt for automationand high throughput analysis.

It will be clear to the person skilled in the art that the acceptorvectors as hereinbefore described can be readily adapted to provide avector which can be used to produce in vitro large amounts of doublestranded RNA or RNAi comprising a complementary sense and antisenseportion essentially similar to a target gene of choice as describedelsewhere in this application, by exchanging the promoter capable ofbeing expressed in a eukaryotic cell for a promoter recognized by anyRNA polymerase. Very suitable promoters to this end are the promotersrecognized by bacteriophage single subunit RNA polymerases such as thepromoters recognized by bacteriophage single subunit RNA polymerase suchas the RNA polymerases derived from the E. coli phages T7, T3, φI, φII,W31, H, Y, A1, 122, cro, C21, C22, and C2; Pseudomonas putida phagegh-1; Salmonella typhimurium phage SP6; Serratia marcescens phage IV;Citrobacter phage ViIII; and Klebsiella phage No.11 (Hausmann, CurrentTopics in Microbiology and Immunology, 75: 77-109 (1976); Korsten etal., J. Gen Virol. 43: 57-73 (1975); Dunn et al., Nature New Biology,230: 94-96 (1971); Towle et al., J. Biol. Chem. 250: 1723-1733 (1975);Butler and Chamberlin, J. Biol. Chem., 257: 5772-5778 (1982)). Examplesof such promoters are a T3 RNA polymerase specific promoter and a T7 RNApolymerase specific promoter, respectively. A T3 promoter to be used asa first promoter in the CIG can be any promoter of the T3 genes asdescribed by McGraw et al, Nucl. Acid Res. 13: 6753-6766 (1985).Alternatively, a T3 promoter may be a T7 promoter that is modified atnucleotide positions-10, -11 and -12 in order to be recognized by T3 RNApolymerase (Klement et al., J. Mol. Biol. 215, 21-29 (1990)). Apreferred T3 promoter is the promoter having the “consensus” sequencefor a T3 promoter, as described in U.S. Pat. No. 5,037,745. A T7promoter which may be used according to the invention, in combinationwith T7 RNA polymerase, comprises a promoter of one of the T7 genes asdescribed by Dunn and Studier, J. Mol. Biol. 166: 477-535 (1983). Apreferred T7 promoter is the promoter having the “consensus” sequencefor a T7 promoter, as described by Dunn and Studier (supra).

Thus, the invention also provides an acceptor vector comprising:

origin of replication allowing replication in a host cell (1);

a selectable marker region (2) capable of being expressed in the hostcell; and

a chimeric DNA construct comprising in sequence:

a promoter or promoter region (3) capable of being recognized by abacteriophage single subunit RNA polymerase;

a first recombination site (4), a second recombination site (5), a thirdrecombination site (6) and a fourth recombination site (7) whereby

the first (4) and fourth recombination site (7) are capable of reactingwith the same other recombination site and preferably are identical toeach other;

the second (5) and third (6) recombination site are also capable ofreacting with the same other recombination site and preferably areidentical to each other;

the first (4) and second (5) recombination site do not recombine witheach other or with the same other recombination site; and

the third (6) and fourth (7) recombination site do not recombine witheach other or with the same other recombination site; and

a 3′ transcription terminating and polyadenylation region (8) functionalin a eukaryotic cell.

The acceptor vector may be used to convert a DNA fragment of interestinto an inverted repeat structure as described elsewhere in theapplication and dsRNA can be produced in large amounts by contacting theacceptor vector DNA with the appropriate bacteriophage single subunitRNA polymerase under conditions well known to the skilled artisan. Theso-produced dsRNA can then be used for delivery into cells prone to genesilencing, such as plant cells, fungal cells or animal cells. dsRNA maybe introduced in animal cells via liposomes or other transfection agents(e.g. Clonfection transfection reagent or the CalPhos Mammaliantransfection kit from ClonTech) and could be used for methods oftreatment of animals, including humans, by silencing the appropriatetarget genes.

The acceptor vectors may also be equipped with any prokaryotic promotersuitable for expression of dsRNA in a particular prokaryotic host. Theprokaryotic host can be used as a source of dsRNA, e.g. by feeding it toan animal, such as a nematode, in which the silencing of the target geneis envisioned.

The promoter capable of expression in eukaryotic cell may also be apromoter capable of expression in a mammalian cell and vectors accordingto the invention may transiently be delivered using a retroviraldelivery system or other animal transfection system.

In another embodiment of the invention, a method is provided for makinga eukaryotic organism, particularly a plant, wherein the phenotypicexpression of a target nucleic acid of interest is reduced or inhibited,comprising the steps of preparing a chimeric DNA construct comprising anucleic acid of interest (12) comprising a nucleotide sequence of atleast 19 bp or 25 bp having at least 70% sequence identity to the targetnucleic acid of interest and capable of expressing a dsRNA in cells ofthe eukaryotic organism, particularly a plant according to the methodsof the current invention and introducing the chimeric DNA construct incells of the eukaryotic organism, and isolating eukaryotic organismtransgenic for the chimeric DNA construct.

As used herein, “phenotypic expression of a target nucleic acid ofinterest” refers to any quantitative trait associated with the molecularexpression of a nucleic acid in a host cell and may thus include thequantity of RNA molecules transcribed or replicated, the quantity ofpost-transcriptionally modified RNA molecules, the quantity oftranslated peptides or proteins, the activity of such peptides orproteins.

A “phenotypic trait” associated with the phenotypic expression of anucleic acid of interest refers to any quantitative or qualitativetrait, including the trait mentioned, as well as the direct or indirecteffect mediated upon the cell, or the organism containing that cell, bythe presence of the RNA molecules, peptide or protein, orposttranslationally modified peptide or protein. The mere presence of anucleic acid in a host cell, is not considered a phenotypic expressionor a phenotypic trait of that nucleic acid, even though it can bequantitatively or qualitatively traced. Examples of direct or indirecteffects mediated on cells or organisms are, e.g., agronomically orindustrial useful traits, such as resistance to a pest or disease;higher or modified oil content etc.

As used herein, “reduction of phenotypic expression” refers to thecomparison of the phenotypic expression of the target nucleic acid ofinterest to the eukaryotic cell in the presence of the RNA or chimericgenes of the invention, to the phenotypic expression of the targetnucleic acid of interest in the absence of the RNA or chimeric genes ofthe invention. The phenotypic expression in the presence of the chimericRNA of the invention should thus be lower than the phenotypic expressionin absence thereof, preferably be only about 25%, particularly onlyabout 10%, more particularly only about 5% of the phenotypic expressionin absence of the chimeric RNA, especially the phenotypic expressionshould be completely inhibited for all practical purposes by thepresence of the chimeric RNA or the chimeric gene encoding such an RNA.

A reduction of phenotypic expression of a nucleic acid where thephenotype is a qualitative trait means that in the presence of thechimeric RNA or gene of the invention, the phenotypic trait switches toa different discrete state when compared to a situation in which suchRNA or gene is absent. A reduction of phenotypic expression of a nucleicacid may thus, i.a. be measured as a reduction in transcription of (partof) that nucleic acid, a reduction in translation of (part of) thatnucleic acid or a reduction in the effect the presence of thetranscribed RNA(s) or translated polypeptide(s) have on the eukaryoticcell or the organism, and will ultimately lead to altered phenotypictraits. It is clear that the reduction in phenotypic expression of atarget nucleic acid of interest, may be accompanied by or correlated toan increase in a phenotypic trait.

As used herein a “target nucleic acid of interest” refers to anyparticular RNA molecule or DNA sequence which may be present in aeukaryotic cell, particularly a plant cell whether it is an endogenousnucleic acid, a transgenic nucleic acid, a viral nucleic acid, or thelike.

Methods for making transgenic eukaryotic organisms, particularly plantsare well known in the art. Gene transfer can be carried out with avector that is a disarmed Ti-plasmid, comprising a chimeric gene of theinvention, and carried by Agrobacterium. This transformation can becarried out using the procedures described, for example, in EP 0 116718. Particular kinds of Agrobacterium mediated transformation methodsare the so-called in planta methods, which are particularly suited forArabidopsis spp. transformation (e.g., Clough and Bent, Plant J. 16:735-534, 1998). Alternatively, any type of vector can be used totransform the plant cell, applying methods such as direct gene transfer(as described, for example, in EP 0 233 247), pollen-mediatedtransformation (as described, for example, in EP 0 270 356, WO85/01856and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation(as described, for example, in EP 0 067 553 and U.S. Pat. No.4,407,956), liposome-mediated transformation (as described, for example,in U.S. Pat. No. 4,536,475), and the like. Other methods, such asmicroprojectile bombardment, as described for corn by Fromm et al.(1990) and Gordon-Kamm et at (1990), are suitable as well. Cells ofmonocotyledonous plants, such as the major cereals, can also betransformed using wounded and/or enzyme-degraded compact embryogenictissue capable of forming compact embryogenic callus, or wounded and/ordegraded immature embryos as described in WO92/09696. The resultingtransformed plant cell can then be used to regenerate a transformedplant in a conventional manner.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the chimeric gene for reduction of the phenotypicexpression of a nucleic acid of interest of the invention in othervarieties of the same or related plant species, or in hybrid plants.Seeds obtained from the transformed plants contain the chimeric genes ofthe invention as a stable genomic insert.

In another embodiment, the invention provides a method for isolating anucleic acid molecule involved in determining a particular phenotypictrait of interest. The method involves the following steps:

-   -   preparing a library of chimeric DNA constructs capable of        expressing a dsRNA in cells of the eukaryotic non-human organism        using the methods and means described in the current invention;    -   introducing individual representatives of this library of        chimeric DNA constructs in cells of the eukaryotic non-human        organism, preferably by stable integration in their genome,        particularly their nuclear genome;    -   isolating a eukaryotic organism exhibiting the particular trait;        and    -   isolating the corresponding nucleic acid molecule present in the        eukaryotic organism with the trait of interest, preferably from        the aforementioned library.

It will be understood that the methods and means of the invention may beused to determine the function of an isolated nucleic acid fragment orsequence with unknown function, by converting a part or the whole ofthat nucleic acid fragment or sequence according to the methods of theinvention into a chimeric construct capable of making a dsRNA transcriptwhen introduced in a eukaryotic cell, introducing that chimeric DNAconstruct into a eukaryotic organism to isolate preferably a number oftransgenic organisms and observing changes in phenotypic traits.

The invention also provides acceptor vectors, as described in thisspecification as well as kits comprising such vectors.

It will be understood that the vectors, methods and kits according tothe invention may be used in all eukaryotic organisms which are prone togene silencing including yeast, fungi, plants, animals such asnematodes, insects and arthropods, vertebrates including mammals andhumans.

Also provided by the invention are non-human organisms comprisingchimeric DNA constructs comprising in sequence the following operablylinked DNA fragments a promoter or promoter region (3) capable of beingrecognized by RNA polymerases of the eukaryotic cell; a recombinationsite (15) which is the recombination product of the first (4)recombination site on the acceptor vector and the fifth recombinationsite (13) flanking the DNA of interest; a first DNA copy of the nucleicacid fragment of interest (12);

a recombination site (16) which is the recombination product of thesecond (4) recombination site on the acceptor vector and the sixthrecombination site (14) flanking the DNA of interest;

a recombination site (17) which is the recombination product of thethird (5) recombination site on the acceptor vector and the sixthrecombination site (14) flanking the DNA of interest;

a second DNA copy of the nucleic acid fragment of interest in oppositeorientation (12) compared to the first copy;

a recombination site (18) which is the recombination product of thefourth (7) recombination site on the acceptor vector and the fifthrecombination site (13) flanking the DNA of interest; and

a 3′ transcription terminating and polyadenylation region (8) functionalin a eukaryotic cell.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA region that is functionally orstructurally defined, may comprise additional DNA regions etc.

The term “gene” means any DNA fragment comprising a DNA region (the“transcribed DNA region”) that is transcribed into a RNA molecule (e.g.,a mRNA) in a cell operably linked to suitable regulatory regions, e.g.,a plant-expressible promoter. A gene may thus comprise several operablylinked DNA fragments such as a promoter, a 5′ leader sequence, a codingregion, and a 3′ region comprising a polyadenylation site. A plant geneendogenous to a particular plant species (endogenous plant gene) is agene which is naturally found in that plant species or which can beintroduced in that plant species by conventional breeding. A chimericgene is any gene that is not normally found in a plant species or,alternatively, any gene in which the promoter is not associated innature with part or all of the transcribed DNA region or with at leastone other regulatory region of the gene.

The term “expression of a gene” refers to the process wherein a DNAregion which is operably linked to appropriate regulatory regions,particularly to a promoter, is transcribed into an RNA which isbiologically active i.e., which is either capable of interaction withanother nucleic acid or which is capable of being translated into apolypeptide or protein. A gene is the to encode an RNA when the endproduct of the expression of the gene is biologically active RNA, suchas e.g. an antisense RNA, a ribozyme or a replicative intermediate. Agene is the to encode a protein when the product of the expression ofthe gene is a protein or polypeptide.

A nucleic acid is “capable of being expressed”, when the nucleic acid,when introduced in a suitable host cell, particularly in a plant cell,can be transcribed (or replicated) to yield an RNA, and/or translated toyield a polypeptide or protein in that host cell.

The following non-limiting Examples describe the construction ofacceptor vectors and the application thereof for the conversion ofnucleic acid fragments of interest into chimeric DNA constructs capableof expressing a dsRNA transcript in eukaryotic cells. Unless statedotherwise in the Examples, all recombinant DNA techniques are carriedout according to standard protocols as described in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel etal. (1994) Current Protocols in Molecular Biology, Current Protocols,USA. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK. Other references for standard molecularbiology techniques include Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, NY, Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for polymerase chain reactions can be found in Dieffenbach andDveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring HarborLaboratory Press, and in McPherson at al. (2000) PCR-Basics: FromBackground to Bench, First Edition, Springer Verlag, Germany.

Throughout the description and Examples, reference is made to thefollowing sequences:

-   SEQ ID No 1: core sequence of recombination site attB1-   SEQ ID No 2: core sequence of recombination site attB2-   SEQ ID No 3: core sequence of recombination site attB3-   SEQ ID No 4: core sequence of recombination site attR1-   SEQ ID No 5: core sequence of recombination site attR2-   SEQ ID No 6: core sequence of recombination site attR3-   SEQ ID No 7: core sequence of recombination site attL1-   SEQ ID No 8: core sequence of recombination site attL2-   SEQ ID No 9: core sequence of recombination site attL3-   SEQ ID No 10: core sequence of recombination site attP1-   SEQ ID No 11: core sequence of recombination sites attP2,P3-   SEQ ID No 12: nucleotide sequence of chalcone synthase gene of    Arabidopsis-   SEQ ID No 13: nucleotide sequence of the acceptor vector    “pHELLSGATE”-   SEQ ID No 14: oligonucleotide attB1 “forward” primer used for    amplification of 400 bp and 200 bp CHS fragments.-   SEQ ID No 15: oligonucleotide attB2 “reverse” primer for    amplification of the 400 bp CHS fragment.-   SEQ ID No 16: oligonucleotide attB2 “reverse” primer for    amplification of the 200 bp CHS fragment.-   SEQ ID No 17: oligonucleotide attB1 “forward” primer used for    amplification of 100 bp CHS fragment.-   SEQ ID No 18: oligonucleotide attB2 “reverse” primer for    amplification of the 100 bp CHS fragment.-   SEQ ID No 19: oligonucleotide attB1 “forward” primer used for    amplification of 50 bp CHS fragment.-   SEQ ID No 20: oligonucleotide attB2 “reverse” primer for    amplification of the 50 bp CHS fragment.-   SEQ ID No 21: oligonucleotide attB1 “forward” primer for    amplification of the 25 bp CHS fragment.-   SEQ ID No 22: oligonucleotide attB2 “reverse” primer for the 25 bp    fragment.-   SEQ ID No 23: nucleotide sequence of the acceptor vector “pHELLSGATE    4”-   SEQ ID No 24: nucleotide sequence of the acceptor vector “pHELLSGATE    8”-   SEQ ID No 25: nucleotide sequence of the acceptor vector “pHELLSGATE    11”-   SEQ ID No 26: nucleotide sequence of the acceptor vector “pHELLSGATE    12”

EXAMPLES Example 1 Construction of the Acceptor Vector pHELLSGATE

With the completion of the Arabidopsis genome project, the advent ofmicro-array technology and the ever-increasing investigation into plantmetabolic, perception, and response pathways, a rapid targeted way ofsilencing genes would be of major assistance. The high incidence anddegree of silencing in plants transformed with chimeric genes containingsimultaneously a sense and antisense nucleotide sequence, as well as afunctional intron sequence suggested that such vectors could form thebasis of a high-throughput silencing vector. However, one of the majorobstacles in using such conventional cloning vectors for a large numberof defined genes or a library of undefined genes would be cloning thehairpin arm sequences for each gene in the correct orientations.

Attempts to clone PCR products of sense and antisense arms together withthe appropriately cut vector as a single step four-fragment ligationfailed to give efficient or reproducible results. Therefore, a construct(PHELLSGATE) was made to take advantage of Gateway™ (Life Technologies).With this technology, a PCR fragment is generated, bordered withrecombination sites (attB1 and attB2) which is directionally recombined,in vitro, into a plasmid containing two sets of suitable recombinationsites (attP1 and attP2 sites) using the commercially availablerecombination protein preparation.

The pHELLSGATE vector was designed such that a single PCR product fromprimers with the appropriate attB1 and attB2 sites would be recombinedinto it simultaneously to form the two arms of the hairpin. The ccdBgene, which is lethal in standard E. coli strains such as DH5α (but notin DB3.1), was placed in the locations to be replaced by the armsequences, ensuring that only recombinants containing both arms would berecovered. Placing a chloramphenicol resistance gene within the intron,gives a selection to ensure the retention of the intron in therecombinant plasmid.

pHELLSGATE comprises the following DNA fragments:

-   -   a spectinomycin/streptomycin resistance gene (SEQ ID No 13 from        the nucleotide at position 7922 to the nucleotide sequence at        9985);    -   a right T-DNA border sequence (SEQ ID No 13 from the nucleotide        at position 10706 to the nucleotide sequence at 11324);

-   a CaMV35S promoter (SEQ ID No 13 from the nucleotide at position    11674 to the nucleotide sequence at 13019);

-   an attP1 recombination site (complement of the nucleotide sequence    of SEQ ID No 13 from the nucleotide at position 17659 to the    nucleotide sequence at 17890);

-   a ccdB selection marker (complement of the nucleotide sequence of    SEQ ID No 13 from the nucleotide at position 16855 to the nucleotide    at position 17610);

-   an attP2 recombination site (complement of the nucleotide sequence    of SEQ ID No 13 from the nucleotide at position 16319 to the    nucleotide at position 16551);

-   pdk intron2 (SEQ ID No 13 from the nucleotide at position 14660 to    the nucleotide at position 16258) flanked by the intron splice site    (TACAG*TT (SEQ ID No 13 from the nucleotide at position 16254 to the    nucleotide sequence at 16260) and the intron splice site (TG*GTAAG)    (SEQ ID No 13 from the nucleotide at position 14660 to the    nucleotide sequence at 14667) and comprising a chloramphenicol    resistance gene (SEQ ID No 13 from the nucleotide at position 15002    to the nucleotide at position 15661);

-   an attP2 recombination site (SEQ ID No 13 from the nucleotide at    position 14387 to the nucleotide at position 14619);

-   a ccdB selection marker (complement of the nucleotide sequence of    SEQ ID No 13 from the nucleotide at position 13675 to the nucleotide    at position 13980);

-   an attP1 recombination site (SEQ ID No 13 from the nucleotide at    position 13048 to the nucleotide at position 13279);

-   an octopine synthase gene terminator region (SEQ ID No 13 from the    nucleotide at position 17922 to the nucleotide sequence at 18687);

-   a chimeric marker selectable in plants comprising:

-   a nopaline synthase promoter (SEQ ID No 13 from the nucleotide at    position 264 to the nucleotide sequence at 496);

-   a nptII coding region (SEQ ID No 13 from the nucleotide at position    497 to the nucleotide sequence at 1442); and

-   a nopaline synthase gene terminator (SEQ ID No 13 from the    nucleotide at position 1443 to the nucleotide sequence at 2148);

-   a left T-DNA border sequence (SEQ ID No 13 from the nucleotide at    position 2149 to the nucleotide sequence at 2706);

-   an origin of replication; and

-   a kanamycin resistance gene;

The complete nucleotide sequence of pHELLSGATE is represented in thesequence listing (SEQ ID No 13) and a schematic figure can be found inFIG. 3.

Example 2 Use of the pHELLSGATE to Convert Nucleic Acid Fragments ofInterest into dsRNA Producing Chimeric Silencing Genes.

To test the acceptor vector pHELLSGATE, about 400 bp, 200 bp, 100 bp, 50bp and 25 bp fragments of the Arabidopsis thaliana chalcone synthaseisomerase coding sequence (SEQ ID No 12) (having respectively thenucleotide sequence of SEQ ID No 12 from the nucleotide at position 83to the nucleotide at position 482; the nucleotide sequence of SEQ ID No12 from the nucleotide at position 83 to the nucleotide at position 222;the nucleotide sequence of SEQ ID No 12 from the nucleotide at position83 to the nucleotide at position 182; the nucleotide sequence of SEQ IDNo 12 from the nucleotide at position 83 to the nucleotide at position132; and the nucleotide sequence of SEQ ID No 12 from the nucleotide atposition 83 to the nucleotide at position 107) were used as nucleic acidfragments of insert for construction of chimeric genes capable ofproducing dsRNA.

This gene was chosen because its mutant allele has been reported inArabidopsis to give distinct phenotypes. The CHS tt4(85) EMS mutant(Koornneef, 1990) produces inactive CHS resulting in no anthocyaninpigment in either the stem or seed-coat. Wildtype plants produce thepurple-red pigment in both tissues.

In a first step, the respective fragments were PCR amplified usingspecific primers further comprising attB1 and attB2 recombination sites.AttB1 and attB2 specific primers were purchased from Life Technologies.The 25 and 50 bp fragments flanked by att sites were made bydimerization of the primers.

The following combinations of primers were used:

For the 400 bp fragment:

Forward Primer:

GGGGACAAGTTTGTACAAAAAAGCAGGCTGCACTGC (SEQ ID No 14)TAACCCTGAGAACCATGTGCTTC;Reverse Primer:

GGGGACCACTTTGTACAAGAAAGCTGGGTCGCTTGA (SEQ ID No 15)CGGAAGGACGGAGACCAAGAAGC.For the 200 bp fragment:Forward Primer:

GGGGACAAGTTTGTACAAAAAAGCAGGCTGCACTGC (SEQ ID No 14)TAACCCTGAGAACCATGTGCTTC;Reverse Primer:

GGGGACCACTTTGTACAAGAAAGCTGGGTAGGAGCC (SEQ ID No 16)ATGTAAGCACACATGTGTGGGTT.For the 100 bp fragment:Forward Primer:

GGGGACAAGTTTGTACAAAAAAGCAGGCTGCACTGC (SEQ ID No 17)TAACCCTGAGAACCATGTGCTTCAGGCGGAGTATCC TGACTACTACTTCCGCATCACCAACAGT;Reverse Primer:

GGGGACCACTTTGTACAAGAAAGCTGGGTAACTTCT (SEQ ID No 18)CCTTGAGGTCGGTCATGTGTTCACTGTTGGTGATGC GGAAGTAGTAGTCAGGATACTCCGCCTG.For the 50 bp fragment:Forward Primer:

GGGGACAAGTTTGTACAAAAAAGCAGGCTGCACTGC (SEQ ID No 19)TAACCCTGAGAACCATGTGCTTCAGGCGGAGTATCC TGACTAC;Reverse Primer:

GGGGACCACTTTGTACAAGAAAGCTGGGTGTAGTCA (SEQ ID No 20)GGATACTCCGCCTGAAGCACATGGTTCTCAGGGTTA GCAGTGC.For the 25 bp fragment:Forward Primer:

GGGGACAAGTTTGTACAAAAAAGCAGGCTGCACTGC (SEQ ID No 21) TAACCCTGAGAACCATGT;Reverse Primer:

GGGGACCACTTTGTACAAGAAAGCTGGGTACATGGT (SEQ ID No 22) TCTCAGGGTTAGCAGTGC.

PCR amplification and recombination using the GATEWAY™ technology withthe commercially available BP Clonase (Life Technologies) were performedaccording to the manufacturer's instructions.

Bacterial colonies obtained on chloramphenicol-containing plates spreadwith E. coli DH5α bacteria, transformed (by electroporation or byheatshocking RbCI2 treated competent E. coli cells) with the in vitrorecombination reaction were screened. Colonies containing the desiredrecombinant plasmid were obtained in each case. For the about 400 bpfragment, 24 colonies were screened and 23 contained the desiredconstruct with the 400 bp in inverted repeat, operably linked to theCaMV35S promoter. For the about 200 bp fragment, 36 colonies werescreened and 35 contained the desired construct with the 200 bp ininverted repeat, operably linked to the CaMV35S promoter. For the about50 bp fragment, six colonies were screened and four contained thedesired construct with the 50 bp in inverted repeat, operably linked tothe CaMV35S promoter. For the 25 bp fragment, six colonies were screenedand one contained the desired construct with the 400 bp in invertedrepeat, operably linked to the CaMV35S promoter. In a number of cases,the structure was confirmed by sequence analysis.

These results show that this vector facilitates the rapid, efficient,and simple production of hpRNA (hairpin RNA constructs). pHELLSGATE is aT-DNA vector, with a high-copy-number origin of replication for ease ofhandling. Recombinant pHELLSGATE constructs can be directly transformedinto Agrobacterium for transformation of the chimeric construct intoplants. This system can be used in high throughput applications.

Example 3 Evaluation of Plants Comprising the Chimeric Genes of Example2.

The vectors containing the dsRNA producing chimeric constructs with the400, 200, 100, 50 and 25 nucleotides of chalcone synthase in invertedrepeat (Example 2) were introduced into Agrobacterium tumefaciens strainAGL1, GV3101 or LBA4404 either by electroporation or tri-parentalmating.

Transgenic Arabidopsis lines are obtained by transformation with theseAgrobacteria using the dipping method of Clough and Bent (1998).

Chalcone synthase activity is monitored by visual observation of stemand leaf color (normally in plants grown under high light, and byunaided or microscope assisted visual observation of seed-coat color.

Most of the transgenic lines transformed with the above-mentioned CHSsilencing constructs show pronounced silencing. The seed color of mostof these lines is virtually indistinguishable from seed of the tt4(85)mutant to the naked eye. Examination of the seed under a lightmicroscope reveals that the degree of pigmentation is generally uniformin the cells of the coat of an individual seed, and among seeds of thesame line.

Example 4 Construction of the Acceptor Vectors pHELLSGATE 4, pHELLSGATE8, pHELLSGATE 11 and pHELLSGATE 12.

pHELLSGATE 4 was made by excising the DNA fragment comprising the pdkintron and chloramphenicol resistance gene from pHellsgate (Example 1)with HindIII and EcoRI and replacing it with a HindIII/EcoRI DNAfragment containing only the pdk intron. The complete nucleotidesequence of pHELLSGATE 4 is represented in the sequence listing (SEQ IDNo 23).

pHellsgate 8 was made by PCR amplification using pHellsgate DNA as atemplate and oligonucleotides with the sequence5′GGGCTCGAGACAAGTTTGTACAAAAMGCTG 3′ and 5′GGCTCGAGACCACTTTGTACAAGAAAGC3′ as primers. These primers modify the attP sites within pHellsgate toattR sites. The resulting fragment was sequenced and inserted into theXhoI site of a vector upstream of a DNA fragment containing the pdkintron fragment. Similarly an XbaI/XbaI fragment amplified with theoligonucleotides 5′GGGTCTAGACAAGTTTGTACAAAAAAGCTG 3′ and5′GGGTCTAGACCACTTTGTACAAGAAAGC 3′ as primers and pHELLSGATE as templateDNA to modify the attP sites of this cassette to attR sites. Thisfragment was sequenced and inserted into the XbaI site of theintermediate described above downstream of the pdk intron. The completenucleotide sequence of pHELLSGATE 8 is represented in the sequencelisting (SEQ ID No 24) and a schematic figure can be found in FIG. 4.

pHELLSGATE 11 is similar to pHELLSGATE 8 except that the pdk intron hasbeen engineered to contain a branching point in the complementary strandsuch that splicing of the intron is independent of its orientation (aso-called “two-way intron”). The complete nucleotide sequence ofpHELLSGATE 11 is represented in the sequence listing (SEQ ID No 25) anda schematic representation thereof can be found in FIG. 4.

pHELLSGATE 12 is also similar to pHELLSGATE 8 except that the pdk intronhas been duplicated as an inverted repeat. The complete nucleotidesequence of pHELLSGATE 12 is represented in the sequence listing (SEQ IDNo 26) and a schematic representation thereof can be found in FIG. 4.

Example 5 Use of the Different pHELLSGATE Vectors to Generate dsRNAChimeric Silencing Genes Targeted Towards Three Different Model TargetGenes.

The efficiency in gene silencing of the different pHELLSGATE vectors wastested by inserting fragments of three target genes: Flowering locus C(FLC); Ethylene insensitive 2 (EIN2); and Phytoene desaturase (PDC). ForFLC a 390 bp fragment was used (from the nucleotide at position 303 tothe nucleotide at position 692 of the nucleotide sequence available asGenbank Accession Nr AF116527). For EIN2 a 580 bp fragment was used(from the nucleotide at position 541 to the nucleotide at position 1120of the nucleotide sequence available as Genbank Accession Nr AF141203).For PDS a 432 bp fragment was used (from the nucleotide at position 1027to the nucleotide at position 1458 of the nucleotide sequence availableas Genbank Accession Nr L16237). Genes of interest were amplified usinggene specific primers with either a 5′ attB1 extension(GGGGACAAGTTTGTACAAAAAAGCAGGCT) or an attB2 extension(GGGACCACTTTGTACAAGAAAGCTGGGT) using F1 Taq DNA polymerase (FisherBiotec, Subiaco, Wash., Australia) according to the manufacturer'sprotocol. PCR products were precipitated by adding 3 volumes TE and twovolumes 30% (w/v) PEG 3000, 30 mM MgCl₂ and centrifuging at 13000 g for15 minutes.

Recombination reaction of PCR products with either pDONR201 (Invitrogen,Groningen, The Netherlands) or pHellsgate 4 were carried out in a totalvolume of 10 μL with 2 μL BP clonase buffer (Invitrogen), 1-2 μL PCRproduct 150 ng plasmid vector and 2 μL BP clonase (Invitrogen). Thereaction was incubated at room temperature (25° C.) for 1 h toovernight. After the incubation, 1 μL proteinase K (2 μg/μL; Invitrogen)was added and incubated for 10 min at 37° C. 1-2 μL of the mix was usedto transform DH5α; colonies were selected on the appropriateantibiotics. Clones were checked either by digestion of DNA minipreps orPCR. Recombination reactions from pDONR201 clones to pHellsgate 8, 11 or12 were carried out in 10 μL total volume with 2 μL LR clonase buffer(Invitrogen), 2 μL pDONR201 clone (approximately 150 ng), 300 ngpHellsgate 8, 11 or 12 and 2 μL LR clonase (Invitrogen). The reactionwas incubated overnight at room temperature, proteinase-treated and usedto transform E. coli DH5α as for the BP clonase reaction.

Transformation of Arabidopsis was performed according to via the floraldip method (Clough and Bent, 1998). Plants were selected on agarsolidified MS media supplemented with 100 mgA timentin and 50 mgAkanamycin. For FLC and PDS constructs, the C24 ecotype was used; forEIN2 constructs, Landsberg erecta was used. For scoring of EIN2phenotypes, transformed T1 plants were transferred to MS mediacontaining 50 μM 1-aminocyclopropane-1-carboxylic acid (ACC) togetherwith homozygous EIN2-silenced lines and wild type Landberg erectaplants. T1 FLC hpRNA plants were scored by transferring to MS plates andscoring days to flower or rosette leaves at flowering compared to C24wild type plants and flc mutant lines. T1 PDS hpRNA plants were scoredby looking at bleaching of the leaves. The results of the analysis ofplants transformed with the different pHELLSGATE vectors are shown inTable 1.

All plants transformed with pHellsgate 4-FLC and pHellsgate 8-FLCflowered significantly earlier than wildtype C24 and in both casesplants flowering with the same number of rosette leaves as the flc-20line (carrying a stable Ds insertion in the first intron of the FLCgene) were observed. There was no clear difference in rosette leaves atflowering between the sets of plants transformed with the pHellsgate4-FLC and pHellsgate 8-FLC constructs.

A difference in the effectiveness of the pHellsgate 4-EIN2 andpHellsgate 8-EIN2 plants was observed. Of 36 transformants forpHG4-EIN2, there were no plants with an observable ACC-resistantphenotype under the conditions used for this experiment, whereas eightof the 11 plants carrying the pHG8-EIN2 transgene showed some degree ofACC-resistance. The extent to which the pHG8-EIN2 plants were resistantto ACC was variable indicating that the severity of silencing variesbetween transformants.

The great majority of plants carrying pHG4-PDS and pHG8-PDS showed aphenotype consistent with the loss of photoprotection due to the absenceof carotenoids. The weakest phenotype was a bleaching of the cotyledons,with the true leaves not bleaching at any stage in the life cycle. Thebleached cotyledon phenotype was only seen in plants transformed withPDS hpRNA constructs; we confirmed that the plants with this phenotypealso contained the PDS hpRNA construct (data not shown) stronglysuggesting that this phenotype is due to PDS silencing and not bleachingfrom the kanamycin selection. Plants transformed with the pHellsgate4-PDS construct gave only this weak bleached cotyledon phenotype. Incontrast, the five of the pHellsgate 8-PDS plants had the weak phenotypeand three showed a stronger phenotype with extensive or completebleaching of the true leaves.

TABLE 1 Construct Test genes T1 plants Rate of silencing HELLSGATE 4 FLC13 12 EIN2 36 0 PDS 12 11 HELLSGATE 8 FLC 6 6 EIN2 11 8 PDS 9 8HELLSGATE 11 FLC 2 2 EIN2 30 11 PDS 11 11 HELLSGATE 11 FLC 8 6(intervening EIN2 region in inverse PDS orientation) HELLSGATE 12 FLC 1311 EIN2 26 12 PDS HELLSGATE 12 FLC 9 8 (intervening EIN2 5 2 region ininverse PDS orientation) CHS

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1. A method for preparing a plant comprising a final chimeric DNA construct, said method comprising: preparing a final chimeric DNA construct capable of expressing a dsRNA of interest in a plant cell; said final chimeric DNA construct of interest comprising a DNA of interest (12) comprising a nucleotide sequence encoding said dsRNA of interest according to a method comprising the steps of combining in vitro a vector, an insert DNA and at least one site specific recombinase protein, said vector comprising the following operably linked DNA fragments: an origin of replication allowing replication in a recipient cell (1), a selectable marker region (2) capable of being expressed in said recipient cell; and a first chimeric DNA construct comprising in sequence: a promoter or promoter region (3) capable of being recognized by RNA polymerases of a plant cell; a first recombination site (4), a second recombination site (5), a third recombination site (6) and a fourth recombination site (7); a 3′ transcription terminating and polyadenylation region (8) functional in said plant cell; wherein said first recombination site (4) and said fourth recombination site (7) are capable of reacting with a same recombination site, and said second recombination site (5) and said third recombination site (6), are capable of reacting with a same recombination site; and wherein said first recombination site (4) and said second recombination site (5) do not recombine with each other or with a same recombination site; or said third recombination site (6) and said fourth recombination site (7) do not recombine with each other or with a same recombination site; said insert DNA comprising said DNA segment of interest (12) flanked by a fifth recombination site (13) which is capable of recombining with said first (4) or fourth (7) recombination site on said vector; and a sixth recombination site (14) which is capable of recombining with said second (5) or third (6) recombination site on said vector; said at least one site specific recombination protein being capable of recombining said first (4) or fourth (7) and said fifth (13) recombination site and said second (5) or third (6) and said sixth (14) recombination site; allowing recombination to occur so as to produce a reaction mixture comprising product DNA molecules, said product DNA molecule comprising in sequence: said promoter or promoter region (3) capable of being recognized by RNA polymerases of said plant cell; a recombination site (15) which is the recombination product of said first (4) and said fifth (13) recombination site; said DNA fragment of interest (12); a recombination site (16) which is the recombination product of said second (4) and said sixth (14) recombination site; a recombination site (17) which is the recombination product of said third (5) and said sixth (14) recombination site; said DNA fragment of interest in opposite orientation (12); a recombination site (18) which is the recombination product of said fourth (7) and said fifth (13) recombination site; and said 3′ transcription terminating and polyadenylation region (8) functional in said plant cell; selecting said product DNA molecules comprising said final chimeric DNA construct; and introducing said final chimeric DNA construct into at least one cell of said plant thereby preparing a plant comprising said final chimeric DNA construct.
 2. A plant obtained through the method of claim
 1. 3. The method according to claim 1, wherein said first (4) and second recombination site (5) flank a second selectable marker gene (10) and said third (6) and fourth recombination site (7) flank a third selectable marker gene (9).
 4. The method according to claim 1 wherein said first chimeric DNA construct comprises a region flanked by intron processing signals (11), functional in said plant cell, located between said second recombination site (5) and said third recombination site (6).
 5. The method according to claim 4 wherein said region flanked by intron processing signals is an intron sequence functional in said plant cell.
 6. The method according to claim 4 wherein said vector further comprises a fourth selectable marker gene (19), located between said second (5) and third (6) rccombination site.
 7. The method according to claim 1, wherein said vector comprises a selectable marker gene selected from the group consisting of an antibiotic resistance gene, a tRNA gene, an auxotrophic marker, a toxic gene, a phenotypic marker, an antisense oligonucleotide; a restriction endonuclease; a restriction endonuclease cleavage site, an enzyme cleavage site, a protein binding site, an a sequence complementary PCR primer.
 8. The method according to claim 1, wherein said wherein said promoter (3) of said vector is a plant-expressible promoter.
 9. The method according to claim 1, wherein said first chimeric DNA construct is flanked by left and right border T-DNA sequences.
 10. The method according to claim 1 wherein said vector further comprises a selectable marker gene capable of being expressed in plant cells located between said left and said right T-DNA border sequences.
 11. The method according to claim 1, wherein said vector of claim 8, further comprises an origin of replication capable of functioning in Agrobacterium sp.
 12. The method according to claim 1, wherein said first (4) and fourth (7) recombination site is attR1 comprising the nucleotide sequence of SEQ ID No 4 and said second (5) and third (6) recombination site is attR2 comprising the nucleotide sequence of SEQ ID No
 5. 13. The method according to claim 1, wherein said first (4) and fourth (7) recombination site is attP1 comprising the nucleotide sequence of SEQ ID No 10 and said second (5) and third (6) recombination site is attP2 comprising the nucleotide sequence of SEQ ID No
 11. 14. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No
 13. 15. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No
 23. 16. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No
 24. 17. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No
 25. 18. The method according to claim 1, wherein said vector comprises the sequence of SEQ ID No
 26. 19. The method according to claim 1, wherein said insert DNA is a linear DNA molecule.
 20. The method according to claim 1, wherein said insert DNA is a circular DNA molecule.
 21. The method according to claim 1, wherein said at least one recombination protein is selected from (i) Int (λ integrase) and IHF (integration host factor) and (ii) Int, Xis (λ excisionase), and IHF. 